As a preliminary note, various publications are referenced throughout this disclosure by Arabic numerals in brackets. The full citation corresponding to each reference number is listed following the detailed description section. In other instances, the particular reference is cited in the text of the specification. In either situation, the disclosures of these publications are herein incorporated by reference in their entireties in order to fully and clearly describe the state of the art to which this invention pertains.
The use of visible and near-infrared (NIR) light in clinical practice is growing rapidly. Compounds absorbing or emitting in the visible, NIR, or long-wavelength (UV-A, >350 nm) region of the electromagnetic spectrum are potentially useful for optical tomographic imaging, endoscopic visualization, and phototherapy. However, a major advantage of biomedical optics lies in its therapeutic potential. Phototherapy has been demonstrated to be a safe and effective procedure for the treatment of various surface lesions, both external and internal. Its efficacy is comparable to that of radiotherapy, but without the harmful radiotoxicity to critical non-target organs.
Phototherapy has been in existence for many centuries and has been used to treat various skin surface ailments. As early as 1400 B.C. in India, plant extracts (psoralens), in combination with sunlight, were used to treat vitiligo. In 1903, Von Tappeiner and Jesionek used eosin as a photosensitizer for the treatment of skin cancer, lupus of the skin, and condylomata of female genitalia. Over the years, the combination of psoralens and ultraviolet A (low-energy) radiation has been used to treat a wide variety of dermatological diseases including psoriasis, parapsoriasis, cutaneous T-cell lymphoma, eczema, vitiligo, areata, and neonatal bilirubinemia. Although the potential of cancer phototherapy has been recognized since early 1900's, systematic studies to demonstrate safety and efficacy began only in 1967 with the treatment of breast carcinoma. Dougherty et al. subsequently conclusively established that long-term cure is possible with photodynamic therapy (PDT). Currently, phototherapeutic methods are also being investigated for the treatment of some cardiovascular disorders such as atherosclerosis and vascular restenosis, for the treatment rheumatoid arthritis, and for the treatment of some inflammatory diseases such as Crohn's disease.
Phototherapeutic procedures require photosensitizers that have high absorptivity. These compounds should preferably be chemically inert, and become activated only upon irradiation with light of an appropriate wavelength. Light-initiated selective tissue injury can be induced when these photosensitizers bind to target tissues, either directly or through attachment to a bioactive carrier. Furthermore, if the photosensitizer is also a chemotherapeutic agent (e.g. anthracycline antitumor agents), then an enhanced therapeutic effect can be attained.
Effective photochemical agents should have the following properties: (a) large molar extinction coefficient; (b) long triplet lifetime; (c) high yield of singlet oxygen and/or other reactive intermediates, viz., free radicals, nitrenes, carbenes, open-shell ionic species such as cabonium ions and the like; (d) efficient energy or electron transfer to cellular components; (e) low tendency to form aggregation in aqueous milieu; (f) efficient and selective targeting of lesions; (g) rapid clearance from blood and non-target tissues; (h) low systemic toxicity; and (i) lack of mutagenicity. Photosensitizers operate via two distinct pathways, termed Types 1 and 2. The type 1 mechanism is shown in the following scheme:
After photoexcitation, the Type 1 mechanism involves direct energy or electron transfer from the photosensitizer to the cellular components, thereby causing cell death. After photoexcitation, the Type 2 mechanism involves distinct steps as shown in the following scheme:
In the first step, singlet oxygen is generated by energy transfer from the triplet excited state of the photosensitizer to the oxygen molecules surrounding the tissues. In the second step, collision of a singlet oxygen with the tissues promotes tissue damage. In both Type 1 and Type 2 mechanisms, the photoreaction proceeds via the lowest triplet state of the photosensitizer. Hence, a relatively long triplet lifetime is required for effective phototherapy. In contrast, for diagnostic imaging purposes, a relatively short triplet lifetime is required to avoid photodamage to the tissue caused by photosensitizers.
The biological basis of tissue injury brought about by tumor phototherapeutic agents has been the subject of intensive study. Various reasonable biochemical mechanisms for tissue damage have been postulated even though the type and number of photosensitizers employed in these studies are relatively small. These biochemical mechanisms are as follows: a) cancer cells upregulate the expression of low density lipoprotein (LDL) receptors, and PDT agents bind to LDL and albumin selectively; (b) porphyrin-like substances are selectively taken up by proliferative neovasculature; (c) tumors often contain an increased number of lipid bodies and are thus able to bind to hydrophobic photosensitizers; (d) a combination of “leaky” tumor vasculature and reduced lymphatic drainage causes porphyrin accumulation; (e) tumor cells may have increased capabilities for phagocytosis or pinocytosis of porphyrin aggregates; (f) tumor associated macrophages may be largely responsible for the concentration of photosensitizers in tumors; and (g) cancer cells may undergo apoptosis induced by photosensitizers. Among these mechanisms, (f) and (g) are the most general and, of these two alternatives, there is a general consensus that (f) is the most likely mechanism by which the phototherapeutic effect of porphyrin-like compounds is induced.
Most of the currently known photosensitizers are commonly referred to as PDT agents and operate via the Type 2 mechanism. For example, Photofrin II, a hematoporphyrin derivative, was approved by the United States Food and Drug Administration for the treatment of bladder, esophageal, and late-stage lung cancers. However, Photofrin II has been shown to have several drawbacks: low molar absorptivity, (ε=3000M−1), low singlet oxygen quantum yield (N=0.1), chemical heterogeneity, aggregation, and prolonged cutaneous photosensitivity. Hence, there has been considerable effort in developing safer and more effective photosensitizers for PDT that exhibit improved light absorbance properties, better clearance, and decreased skin photosensitivity compared to those of Photofrin II. These photosensitizers include monomeric porphyrin derivatives, corrins, cyanines, phthalocyanines, phenothiazines, rhodamines, hypocrellins, and the like. However, these phototherapeutic agents also mainly operate via the Type 2 mechanism.
Surprisingly, there has not been much attention directed at developing Type 1 phototherapeutic agents, despite the fact that the Type 1 mechanism seems inherently more efficient than the Type 2 mechanism. First, unlike Type 2, Type 1 photosensitizers do not require oxygen for causing cellular injury. Second, the Type 1 mechanism involves two steps (photoexcitation and direct energy transfer) whereas the Type 2 mechanism involves three steps (photoexcitation, singlet oxygen generation, and energy transfer). Furthermore, some tumors have hypoxic regions that render the Type 2 mechanism ineffective. In spite of the drawbacks associated with the Type 2 mechanism, however, only a small number of compounds have been developed that operate through the Type 1 mechanism, e.g. anthracyline antitumor agents.
Thus, there is a need to develop effective phototherapeutic agents that operate through the Type 1 mechanism. Phototherapeutic efficacy can be further enhanced if the excited state photosensitizers can generate reactive intermediates such as free radicals, nitrenes, carbenes, and the like. These have much longer lifetimes than the excited chromophore and have been shown to cause considerable cell injury.
Targeted delivery of diagnostic and therapeutic agents (generally referred to as ‘haptens,’ ‘effectors,’ or ‘functional units’) such as fluorophores, photosensitzers, radionuclides, paramagnetic agents, and the like to a particular site in the body continues to be of considerable demand in diagnosis, prognosis, and therapy of various lesions [1-4]. The conventional targeting method (referred to as ‘bioconjugate approach’ or ‘pendant design’) involves chemical attachment of these agents to bioactive carriers. Bioactive carriers include small molecule drugs, hormones, peptidomimetics, and the like, as well as macromolecular proteins, polysaccharides, polynucleotides, and the like. The bioconjugate approach has been explored extensively over the past several decades, and has met with moderate success, particularly in tumor detection, when medium and large size carriers (c.a. molecular weight >1000 Daltons) are employed [2, 3]. This resulting moderate success is because attachment of the dyes, drugs, metal complexes, or other effector molecules to macromolecular carriers such as antibodies, antibody fragments, or large peptides does not greatly alter the targeting properties; i.e. the bioconjugate is still able to bind to the receptor effectively. This approach, however, is limited because the diffusion of high molecular weight bioconjugates to tumor cells is highly unfavorable, and is further complicated by the net positive pressure in solid tumors [5]. Furthermore, many dyes in general, tend to form aggregates in aqueous media that lead to fluorescence quenching. Therefore, there is a need to prepare photoactive small molecules that are not only intrinsically useful for biomedical non-medical optical applications, but also are capable of attachment to suitable bioactive molecules.
Accordingly, a need remains for new small molecule dyes capable of absorbing and emanating spectral energy in the visible and/or near infrared spectrum. Pyrazines are a class of photostable small molecules having highly desirable photophysical properties useful for biomedical applications.

Pyrazine derivatives containing electron withdrawing groups at the 2,5 positions and electron donating groups at the 3,6 positions such as 3,6-diamino-2,5-pyrazine-dicarboxylic acid (structure A) and the corresponding amides strongly absorb and emit in the blue to orange regions with a large Stokes shift on the order of ˜100 nm and with fluorescence quantum yields of about 0.4 [6,7] Conversion of the carboxyl group in 1 to the secondary amide derivatives (structure B) produces a bathochromic shift of about 40 nm, and alkylation of the amino group (structure C) results in further red shift of about 40 nm. Hence, the pyrazine scaffold presents an attractive opportunity to ‘tune’ the electronic properties.